PROJECT SUMMARY/ABSTRACT Corneal diseases have a significant impact on the quality of life of patients and also constitute a major problem for health care system. Corneal diseases such as limbal stem cell deficiency (LSCD) and diabetic keratopathy, associated with limbal epithelial stem cell (LESC) dysfunction/loss, may lead to epithelial defects, conjunctivalization, recurrent epithelial erosions, abnormal wound repair and altered vision. Therefore, mechanisms of LESC function in normal and diseased corneal homeostasis and wound healing process could be key to understanding various corneal diseases. The important part of the stem cell niche functioning is interaction between stem cells and their neighboring stromal cells. LESC are in close contact with the cells of the underlying limbal stroma and the vasculature surrounding limbal crypts. It is well established that there is a crosstalk between stromal cells, or keratocytes, and limbal epithelial cells (LECs) through their secreted factors, which are essential for the stem cell maintenance. Any damage to LESC or limbal stromal niche due to the external insults or diseases such as diabetes may lead to pathological state of altered vision and, in severe cases of LESC loss, results in limbal stem cell deficiency and blindness. Therapeutic potentials of extracellular vesicles (EVs) released from various cell type into extracellular space have been widely reported. EVs are nano-sized vesicles containing mRNA, miRNA, DNA, and protein cargo and mediating physiological intercellular crosstalk. They play important roles in intercellular communication and in stem cell maintenance and activation. We propose to investigate limbal EV contribution to corneal regeneration and wound healing, and examine possible differences in EV cargos released from normal and diabetic limbal stromal cells (LSCs) and LECs. We show that normal LSC-derived EVs (LSC-EVs) significantly promote epithelial healing in wounded organ-cultured human corneas. In addition, we found that there is a difference in cargos of EVs derived from normal and diabetic LSC. We hypothesize that limbal resident cell-derived EVs contribute to limbal epithelial and stromal homeostasis. We predict that the unique complement of EV cargos mediates corneal regeneration and wound healing in normal and diseased conditions. The goal of this proposal is to investigate the major mechanisms of action of both human LSC- and LEC-EVs, which are taken up by neighboring cells, in stem cell maintenance or survival and wound repair in cell cultures and in normal and diabetic human organ-cultured corneas. Techniques to be used include, EV isolation, miRNA isolation, next generation sequencing, proteomics, transmission electron and confocal microscopy, and trans-well co-culture system. Relevance to Public Health: investigation of molecular changes in diabetic corneas is essential for understanding of underlying causes and identification of therapeutic targets for diseased cornea and wound healing process in different layers of the cornea.